ASTM E3286-21 PDF
Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment Technologies
Standard Practice for Preparation Of Cell Monolayers on Glass Surfaces for Evaluation of Microbicidal Properties of Non-Chemical Based Antimicrobial Treatment Technologies
- Статус документа:
- Действующий
- Формат:
- Электронный (PDF)
- Количество страниц:
- 7
- Дата публикации:
- 1 октября 2021 г.
- Издание:
- E3286
- ICS:
- 07.100.01
SIGNIFICANCE AND USE 5.1 There are no reproducible standardized protocols for preparing specimens used to evaluate the microbicidal efficacy of non-chemical treatments such as ultraviolet (UV), highenergy electron beam, or other forms of non-chemical antimicrobial technologies. 5.2 Conventional protocols for applying bioburdens to carriers (see Test Method E2197) cause cells to stack upon one another, thereby creating multiple cell layers in which cells in layers closer to the carrier are masked by cells in overlying layers, which makes relative comparison of different non-chemical antimicrobial treatments more difficult. 5.3 Steel and other metal carriers have asperities that can shield a percentage of the applied cells from direct exposure to electromagnetic irradiation. 5.4 The combined effects of 5.2 and 5.3 confound determination of the microbicidal effect of electromagnetic irradiation on test specimens. 5.5 The practice addresses these two confounding factors by: 5.5.1 Using glass microscope slides – the surfaces of which are asperity-free – as carriers. 5.5.2 Reliably depositing bacterial cells onto the carrier as a monolayer. 5.6 The resulting specimen ensures that all microbes deposited onto the carrier are exposed equally to the irradiation source thereby ensuring that the only variables are the controlled ones – starting inoculum concentration, wavelength (λ – in nm), exposure time(s), and resulting energy dose (J). SCOPE 1.1 This practice provides a protocol for creating bacterial cell monolayers on a flat surface. 1.2 The cultures used and culture preparation steps in this Practice are similar to AOAC Method 961.02 and US EPA MB-06. However, test bacteria are applied to the carrier using an automated deposition device (6.2) rather than as a suspension droplet. 1.3 The carrier inspection protocol is similar to US EPA MB-03 except that carrier surfaces are inspected microscopically rather than visually, unaided. 1.4 A monolayer of cells eliminates the confounding effect caused by the shadowing effect of outer layers of bacteria stacked upon other bacteria on test specimens – thereby attenuating directed energy beams (that is, ultraviolet light, high-energy electron beams) before they can reach underlying cells. 1.5 An asperity-free surface eliminates the shadowing effect of specimen surface topology that can block direct exposure of target bacteria to non-chemical antimicrobial treatments. 1.6 This practice provides a reproducible target microbe and surface specimen to minimize specimen variability within and between testing facilities. This facilitates direct data comparisons among various non-chemical antimicrobial technologies. 1.6.1 Antimicrobial pesticides used in clinical and industrial applications are expected to overcome shadowing effects. However, this practice meets a need for a protocol that facilitates relative comparisons among non-chemical antimicrobial treatments. 1.6.2 This practice is not intended to satisfy or replace existing test requirements for liquid chemical antimicrobial treatments (for example Test Methods E1153 and E2197) or established regulatory agency performance standards such as US EPA MB-06. 1.7 This practice was validated using Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442) using a protocol based on AOAC Method 961.02. If other cultures are used, the suitability of this practice must be confirmed by inspecting prepared surfaces, by using scanning electron microscopy (SEM) or comparable high-resolution microscopy. 1.8 The specimens prepared in accordance with this practice are not meant to simulate end-use conditions. 1.8.1 Non-chemical technologies are only to be used on visibly clean, non-porous surfaces. Consequently, a soil load is not used. 1.9 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard. 1.10 Th...
Abstract
Overview
ASTM E3286-21 sets forth a standardized protocol for preparing bacterial cell monolayers on glass surfaces to support the evaluation of microbicidal properties of non-chemical-based antimicrobial treatment technologies. Developed by ASTM International, this standard addresses critical issues encountered in conventional specimen preparation-such as cell stacking and substrate surface irregularities-that can confound test results for ultraviolet (UV), high-energy electron beams, and other non-chemical disinfection modalities.
By utilizing asperity-free glass microscope slides and reliable monolayer cell deposition, ASTM E3286-21 ensures consistent and reproducible exposure of microbes to test conditions. This enables meaningful, direct comparisons among various non-chemical antimicrobial technologies and minimizes technical variability between laboratories.
Key Topics
- Reproducible specimen preparation: Provides a detailed, reproducible protocol for creating uniform bacterial monolayers on smooth glass slides, mitigating variability in test outcomes.
- Minimized confounding factors: Avoids surface shadowing or cell stacking, both of which can shield bacteria from directed energy treatments, by ensuring all cells are equally exposed.
- Applicability to non-chemical antimicrobials: Specifically designed for evaluating the microbicidal efficacy of treatments such as UV light, high-energy electron beams, and other forms of electromagnetic irradiation.
- Automated deposition: Specifies use of an automated inoculum deposition system over traditional manual droplet methods to achieve consistent monolayer formation.
- Microscopic inspection: Mandates microscopic (not visual) inspection of prepared carriers to confirm monolayer integrity, following protocols similar to US EPA MB-03.
- Validated with key organisms: Successfully validated using Staphylococcus aureus and Pseudomonas aeruginosa, with applicability to other cultures contingent on microscopic confirmation of monolayer quality.
Applications
ASTM E3286-21 is valuable in laboratory and industrial contexts where non-chemical antimicrobial technologies must be assessed reliably and compared across platforms. Principal applications include:
- Testing of UV and light-based disinfectants: Offers a robust means to evaluate how effectively such treatments inactivate microorganisms without the influence of substrate shadowing or cell clumping.
- Benchmarking new non-chemical disinfection technologies: Applies a uniform protocol that helps stakeholders compare efficacy data meaningfully.
- Research and regulatory support: Provides standardized data supporting regulatory filings and acceptance where direct comparison with legacy chemical-based methods is not practical.
- Quality assurance in healthcare, pharmaceutical, and cleanroom settings: Ensures replicable specimen preparation, critical for high-stakes antimicrobial testing and validation.
- Education and training: Establishes clear technical instructions for microbiology and materials science laboratories.
This practice is not intended for chemical disinfectant efficacy evaluation or to replace established performance standards, such as those for liquid antimicrobials or specific regulatory requirements (e.g., US EPA MB-06).
Related Standards
Users of ASTM E3286-21 may find the following related standards useful for comprehensive antimicrobial efficacy testing workflows:
- ASTM E2197: Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
- ASTM E1153: Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous Non-Food Contact Surfaces
- AOAC Method 961.02: Germicidal spray products as disinfectants
- US EPA MB-03 & MB-06: Procedures for carrier preparation and germicidal spray testing
- ASTM E2756: Terminology Relating to Antimicrobial and Antiviral Agents
Keywords: ASTM E3286-21, cell monolayer, glass slides, antimicrobial efficacy testing, non-chemical disinfectant, UV disinfection standard, automated inoculum deposition, microbiology standard, laboratory protocol, healthcare disinfection testing
Технические детали
- Технический комитет
- E35 - Pesticides, Antimicrobials, and Alternative Control Agents
- SKU
- ASTM E3286-21
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